1. Field of the Invention
Embodiments of the present invention relate in general to compositions and methods for inhibiting the expression of the angiogenin gene thereby reducing the effects of angiogenin. Embodiments of the present invention also relate to inhibition of angiogenin gene expression by antisense technologies including, but not limited to, the use of antisense oligodeoxynucleotides and their derivatives. Embodiments of the present invention are further directed to compositions and methods for detecting the angiogenin gene, as well as the detection and diagnosis of abnormal expression of the angiogenin gene in cells and tissues. Embodiments of the present invention are also directed to methods for inhibiting metastasis of cells, such as human tumor cells. Furthermore, this invention is directed to treatment of conditions associated with abnormal angiogenesis, including cancer.
2. Description of Related Art
Angiogenin is a potent inducer of angiogenesis [Fett, J. W., Strydom, D. J., Lobb, R. R., Alderman, E. M., Bethune, J. L., Riordan, J F., and Vallee, B. L. (1985) Biochemistry 24, 5480-5486], a complex process of blood vessel formation that consists of several separate but interconnected steps at the cellular and biochemical level: (i) activation of endothelial cells by the action of an angiogenic stimulus, (ii) adhesion and invasion of activated endothelial cells into the surrounding tissues and migration toward the source of the angiogenic stimulus, and (iii) proliferation and differentiation of endothelial cells to form a new microvasculature [Folkman, J., and Shing, Y. (1992) J. Biol. Chem. 267, 10931-10934; Moscatelli, D., and Rifikin, D. B. (1988) Biochim. Biophys. Acta 948,67-85]. Angiogenin has been demonstrated to induce most of the individual events in the process of angiogenesis including binding to endothelial cells [Badet, J., Soncin, F. Guitton, J. D., Lamare, O., Cartwright, T., and Barritault, D. (1989) Proc. NatL. Acad. Sci. U.S.A. 86, 8427-8431], stimulating second messengers [Bicknell, R., and Vallee, B. L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5961-5965], mediating cell adhesion [Soncin, F. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2232-2236], activating cell-associated proteases [Hu, G-F., and Riordan, J. F. (1993) Biochem. Biophys. Res. Commun. 197, 682-687], inducing cell invasion [Hu, G-F., Riordan, J. F., and Vallee, B. L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12096-12100], inducing proliferation of endothelial cells [Hu, G-F., Riordan, J. F., and Vallee, B. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2204-2209] and organizing the formation of tubular structures from the cultured endothelial cells [Jimi, S-I., Ito, K-I, Kohno, K., Ono, M., Kuwano, M., Itagaki, Y., and Isikawa, H. (1995) Biochem. Biophys. Res. Commun. 211, 476-483]. Angiogenin has also been shown to undergo nuclear translocation in endothelial cells via receptor-mediated endocytosis [Moroianu, J., and Riordan, J. F. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1677-1681] and nuclear localization sequence-assisted nuclear import [Moroianu, J., and Riordan, J. F. (1994) Biochem. Biophys. Res. Commun. 203, 1765-1772].
Although originally isolated from medium conditioned by human colon cancer cells (Fett et al., 1985, supra) and subsequently shown to be produced by several other histologic types of human tumors [Rybak, S. M., Fett, J. W., Yao, Q-Z., and Vallee, B. L. (1987) Biochem. Biophys. Res. Commun. 146, 1240-1248; Olson, K. A., Fett, J. W., French, T. C., Key, M. E., and Vallee, B. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 442-446], angiogenin also is a constituent of human plasma and normally circulates at a concentration of 250 to 360 ng/ml [Shimoyama, S., Gansauge, F., Gansauge, S., Negri, G., Oohara, T., and Beger, H. G. (1996) Cancer Res. 56, 2703-2706; Blaser, J., Triebl, S., Kopp, C., and Tschesche, H. (1993) Eur. J. Clin. Chem. Clin. Biochem. 31, 513-516].
While angiogenesis is a tightly controlled process under usual physiological conditions, abnormal angiogenesis can have devastating consequences as in pathological conditions such as arthritis, diabetic retinopathy and tumor growth. It is now well-established that the growth of virtually all solid tumors is angiogenesis dependent [Folkman, J. (1989) J. Natl. Cancer Inst. 82, 4-6]. Angiogenesis is also a prerequisite for the development of metastasis since it provides the means whereby tumor cells disseminate from the original primary tumor and establish at distant sites [Mahadevan, V., and Hart, I. R. (1990) Rev. Oncol. 3, 97-103; Blood C. H., and Zetter B. R. (1990) Biochim. Biophys. Acta 1032, 89-118]. Therefore, interference with the process of tumor-induced angiogenesis should be an effective therapy for both primary and metastatic cancers.
Several inhibitors of the functions of angiogenin have been developed. These include: (i) monoclonal antibodies (mAbs) [Fett, J. W., Olson, K. A., and Rybak, S. M. (1994) Biochemistry 33, 5421-5427], (ii) an angiogenin-binding protein [Hu, G-F, Chang, S-I, Riordan J. F., and Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2227-2231; Hu, G-F., Strydom, D. J., Fett, J. W., Riordan, J. F., and Vallee B. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,1217-1221; Moroianu, J., Fett, J. W., Riordan, J. F., and Vallee B. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3815-3819], (iii) the placental ribonuclease inhibitor (PRI) [Shapiro, R., and Vallee, B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2238-2241], (iv) peptides synthesized based on the C-terminal sequence of angiogenin [Rybak, S. M., Auld, D. S., St. Clair, D. K., Yao, Q-Z., and Fett, J. W. (1989) Biochem. Biophys. Res. Commun. 162, 535-543], and (v) inhibitory site-directed mutants of angiogenin [Shapiro, R., and Vallee, B. L. (1989) Biochemistry 28, 7401-7408]. All inhibit angiogenin's activities but are not directly cytotoxic to human tumor cells grown in tissue culture.
mAbs or the angiogenin-binding protein when administered locally into xenografts of human tumor cells that were injected subcutaneously (s.c.) into athymic mice are able to delay or, remarkedly, completely prevent the appearance of colon, lung and fibrosarcoma tumors in these animals [Olson et al., 1995, supra, Olson, K. A., French, T. C., Vallee, B. L., and Fett, J. W. (1994) Cancer Res. 54, 4576-4579]. Histological examination revealed that the mechanism of tumor growth inhibition was via an anti-angiogenesis mechanism (Olson et al., 1995, supra). Thus, the inactivation of tumor-produced angiogenin or inhibition of expression of the angiogenin gene by tumor cells promise to be a powerful means of managing cancer, either alone or in combination with more conventional therapies (i.e., chemotherapy, radiotherapy, immunotherapy, etc.).
Expression of specific genes may be suppressed by oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the target gene thereby selectively impeding translation and has been termed an "antisense" methodology. In addition, "antigene" or "triplex" methodologies may also suppress expression of genes by using an oligonucleotide which is complementary to a selected site of double stranded DNA thereby forming a triple-stranded complex to selectively inhibit transcription of the gene. Both "antisense" and "antigene" methodologies find utility as molecular tools for genetic analysis. Antisense oligonucleotides have been extensively used to inhibit gene expression in normal and abnormal cells in studies of the function of various cell proteins. Major advances have been made in the development of antisense or antigene reagents for the treatment of disease states in animals and humans ["Antisense Therapeutics" Agrawal, S. (ed.), Humana Press, 1996; Crooke, S. T., and Bennett, C. F. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 107-129; "Prospects for the Therapeutic Use of Antigene Oligonucleotides", Maher, L. J. (1996) Cancer Investigation 14(1), 66-82 each hereby incorporated by reference in its entirety].
As therapeutics, oligonucleotides possess two major requirements for successful drug design--specificity and affinity. These are achieved by selectively targeting particular DNA or RNA sequences exploiting Watson-Crick base pairing with resulting interference of protein production whether through inhibition of gene transcription or translation of mRNA. This approach allows for rapid identification of lead compounds based on knowledge of a relevant gene target species. Recently, improvements have been made in increasing both the stability and affinity of these compounds. Phosphorothioate analogs of oligodeoxynucleotides (ODNs), in which nonbridging phosphoryl oxygens in the backbone of DNA are substituted with sulfur, abbreviated [S]ODNs, are substantially more stable than their native phosphodiester counterparts, while other derivatives, such as those alkylated on sugar oxygen groups, show enhanced target affinity. [S]ODNs possess good biological activity, pharmacology, pharmacokinetics and safety in vivo (Agrawal, 1996, supra, and references therein) and have been used successfully for anti-tumor therapy in animal models (Crooke and Bennett, 1996, supra). Antisense reagents are now in clinical trials for treatment of cancers and viral infections (Agrawal, 1996, supra). Successful inhibition of specific gene function has been achieved by targeting various sites on specific mRNA sequences that include the AUG translational initiation codon, 5'-transcriptional start site, 3'-termination codon and sequences in both the 5'- and 3'-untranslated regions. Experience to date has indicated that success has been achieved by targeting these and other regions.
As examples, U.S. Pat. No. 5,098,890 is directed to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapies for certain cancerous conditions. U.S. Pat. No. 5,135,917 provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression. U.S. Pat. No. 5,087,617 provides methods for treating cancer patients with antisense oligonucleotides. U.S. Pat. No. 5,166,195 provides oligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810 provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication. U.S Pat. No. 5,194,428 provides antisense oligonucleotides having antiviral activity against influenzavirus. U.S. Pat. No. 4,806,463 provides antisense oligonucleotides and methods using them to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717 is directed to a mixed linkage oligonucleotide phosphorothioates complementary to an oncogene. U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells. The nucleic acid sequence of the entire angiogenin gene including the 5'- and 3'-flanking regions has been determined [Kurachi, K., Davie, E. W., Strydom, D. J. Riordan, J. F. and Vallee, B. L. (1985) Biochemistry 24, 5494-5499 hereby incorporated by reference in its entirety]. The native DNA segment coding for angiogenin, as all such mammalian DNA strands, has two strands; a sense strand and an antisense strand held together by hydrogen bonding. The messenger RNA coding for angiogenin has the same nucleotide sequence as the sense strand except that the DNA thymidine is replaced by uridine. Thus, synthetic antisense nucleotide sequences should bind with the DNA and RNA coding for angiogenin.
However, it is unknown whether antisense reagents will in fact be effective for inhibition of angiogenin expression. To date, no oligonucleotide antisense reagents have been designed or demonstrated to be useful in the inhibition of the expression of angiogenin. Accordingly, a need exists to discover oligonucleotide antisense reagents which can prove useful in modulating or inhibiting the expression of angiogenin and to further discover methods by which such oligonucleotide antisense reagents can be used in methods of diagnosis and treatment.